Specific Unbinding Forces Between Mutated Human P-Selectin

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Letter

Specific Unbinding Forces Between Mutated Human P-Selectin Glycoprotein Ligand-1 and Viral Protein-1 Measured Using Force Spectroscopy Chen-Chu Wang, Siva Shanmugan, Chung-Ku Chen, JianRen Hong, Wei-I Sung, Jiunn-Der Liao, and Yuh-Shyong Yang J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b02373 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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The Journal of Physical Chemistry Letters

Specific Unbinding Forces Between Mutated Human P-selectin Glycoprotein Ligand-1 and Viral Protein-1 Measured Using Force Spectroscopy Chen-Chu Wang1, Kundan Sivashanmugan2, Chung-Ku Chen2, Jian-Ren Hong1, Wei-I Sung2, Jiunn-Der Liao2, Yuh-Shyong Yang1,* 1

Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan

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Department of Materials Science and Engineering, National Cheng Kung University, Tainan, Taiwan

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Corresponding author: Prof. Yuh-Shyong Yang, Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30050, Taiwan Fax: 886-3-572-9288 E-mail: [email protected]

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Abstract Protein tyrosine sulfation (PTS) is a key modulator of extracellular protein–protein interaction (PPI), which regulates principal biological processes. For example, the capsid protein VP1 of enterovirus 71 (EV71) specifically interacts with sulfated P-selectin glycoprotein ligand-1 (PSGL-

1) to facilitate virus invasion. Currently available methods cannot be used to directly observe PTS-induced PPI. In this study, atomic force microscopy was used to measure the interaction between sulfated or mutated PSGL-1 and VP1. We found that the binding strength increased by 6.7-fold following PTS treatment on PSGL-1 with a specific anti-sulfotyrosine antibody. Similar results were obtained when the anti-sulfotyrosine antibody was replaced with the VP1 protein of EV71; however, the interaction forces of VP1 were only approximately one-third of those of the anti-sulfotyrosine antibody. We also found that PTS on the tyrosine-51 residue of glutathione Stransferases fusion-PSGL-1 was mainly responsible for the PTS-induced PPI. Our results contribute to the fundamental understanding of PPI regulated through PTS. TOC Graphic

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Posttranslational modifications (PTMs) refer to the covalent and generally enzymatic modifications of proteins during or after protein biosynthesis, which are essential mechanisms exhibited by eukaryotic cells to diversify their protein functions and dynamically coordinate their signalling networks. Protein tyrosine sulfation (PTS) is one of the most common PTMs first reported in 1954.1 PTS occurs in the trans-Golgi network, and sulfated proteins or peptides are generally transmembrane or secretory proteins.2 Tyrosylprotein sulfotransferase (TPST, EC 2.8.2.20) catalyzes the transfer of the sulfuryl group from 3 ′ -phosphoadenosine-5 ′ phosphosulfate onto a specific tyrosine residue within the target proteins widespread in multicellular eukaryotic organisms.3-4 PTS, which can recognize the sulfate group, has been identified as a key modulator of extracellular protein–protein interactions (PPIs), which are involved in hormonal regulation, hemostasis, inflammation, and infectious diseases.5-7 PPIs are involved in a wide range of biological processes, including cell–cell interactions and metabolic and developmental control.8,9 PTS is believed to alter the strength of PPIs and further modulate ligand–receptor binding, intercellular communication, and signalling2,

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; moreover, it has

become a target for drug design.12 For example, sulfated chemokine receptor (CCR5) interacts with the human immunodeficiency virus-1 (HIV-1) gp120, which facilitates the entry of HIV-1 into the cell. Mutation of the four tyrosine residues in CCR5 to phenylalanine reduces HIV infection by 50%–75%.13 In leukocyte adhesion and inflammatory responses, the extreme amino terminus of P-selectin glycoprotein ligand-1 (PSGL-1) carries three potential tyrosine residue sulfation sites (at positions 46, 48, and 51). The tyrosine sulfate esters and glycans on PSGL-1 are the key binding determinants for P-selectin.13 The binding between PSGL-1 of leukocytes and P-selectin of endothelial cells is essential for leukocyte adhesion in the inflammatory response.14 Treatment of 3



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PSGL-1 with arylsulfatase releases the sulfate group from tyrosine residues, thereby reducing its binding ability to P-selectin.15 This observation was confirmed and supported by the point mutagenesis of the potentially sulfated tyrosine residues.16 Enterovirus 71 (EV71) infection is critically associated with PTS on the N-terminal tyrosine residues of PSGL-1, which facilitates the binding of specific strains of EV71 through its capsid protein (VP1).17 Infections of EV71PB (PSGL-1-binding) strains results in encephalitis (fatal) and hand-foot-mouth disease.18 The presence of multiple PTS sites in a single protein, such as in CCR5 and PSGL-1, is common.18 However, the clear identification of the specific PTS site in a PPI event is difficult, and the binding strength of PTS-induced PPI remains unknown. Table S1 presents various genetic, biochemical, and physical methods that have been used for PPI analysis. Such interactive forces have historically been difficult to observe because the infinitesimal interactions result from multiple weak, noncovalent bonds that are formed between the defined portions of the interacting molecular partners. Atomic force microscopy (AFM)19 allows the measurement of interaction forces between atoms attached to the end of the probe and atoms in a specimen to evaluate the weak, noncovalent, usually short-range forces involved in molecular recognition reactions. Self-assembled monolayers (SAMs) have been applied to immobilize samples onto silicon surfaces and probes. The AFM method can be used to measure PTM-induced PPIs for a specific and infinitesimal single molecular bonding force in vitro.20 The present study is the first to use AFM to measure PTS-induced PPIs, and the working principle is presented in Figure 1.

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Figure 1. Principle of AFM measurement of PTS-induced PPIs. A typical force curve and cantilever behavior based on PTS-induced PPI measurement are shown. At positions 1 and 2, the probe approaches and then contacts the silicon surface (blue arrow and line), which generated PPI force. At position 3, the cantilever bends until the force reaches a specified limit and then deflects upward (gray bar). The probe is then withdrawn toward positions 4 and 5 (red arrows). At position 4, the PPI force causes the cantilever to deflect downward (gray bar). The force curve is denoted by a red dotted line. If the cantilever deflection is constant and no PPI force is produced, the force curve follows the yellow dotted line. Finally, at position 5, with further retraction, the PPI force disappears and the probe detaches from the surface. Then, the cantilever returns to its resting position and is ready for another measurement cycle. The bend change of

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the cantilever induces a force curve change (∆d), which is the unbinding force. The PPI force is calculated based on Hooke’s law. The N-terminal tyrosine residues are the most important sites for TPST catalysis and EV-71 PB infection.3,

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Although the interaction between TPST and PSGL-1 is well known at the

biological level, the detailed molecular interactions remain to be elucidated. Previous studies have focused on the difference in TPST activity between the three tyrosine residues and the resulting biological activities. The present study investigated the variation in PTS-induced PPIs at the molecular level to elucidate the exact interaction location and the resulting forces. To rapidly obtain substrates of high purity, tyrosine substrates were purified using the glutathione Stransferase (GST) fusion tag. Although other tyrosine residues existed on the GST fusion tag, the PTS level was negligible and the TPST activity on GST-PSGL-1 was not affected by the GST fusion tag. The GST fusion tag could be an excellent tool for the purification of other types of PTS substrates.16 As shown in Figure S1, we designed recombinant GST-PSGL-1 and mutants that were further purified for the PTS-induced PPI study. Tyrosine residues at positions 46, 48, and 51 were sequentially mutated to phenylalanine [Figure S1(a)]. The enzymes used for PTS were composed of the NusA-tag fusion protein (60 kDa) and TPST (36 kDa).16 The total molecular weight of TPST is 96 kDa. GST-PSGL-1, composed of the GST fusion tag and the PTS fragment of PSGL-1 (ATEYEYLDYDFL), has a molecular weight of 27.8 kDa. VP1, synthesized from a sequence of the EV71 PB strain, has a molecular weight of 33 kDa. Figure S1(b) presents the sodium dodecyl sulfate polyacrylamide gel electrophoresis results of these proteins, indicating their high purity.

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The substrate and probe were treated with H2SO4/H2O2, (3-aminopropyl)triethoxysilane (APTES), glutaraldehyde, GST-PSGL-1, and PTS reaction, sequentially, as shown in Figure S2. The results of the SAM surface chemical states are presented in Figure 2(a). In the first step, H2SO4/H2O2 was used for cleaning and oxidizing the silicon substrate. The O 1s spectrum revealed a SiOx signal with a peak at 532.8 eV (11). Some remaining oxides on the silicon surface produce O 1s 532.5 eV (10) and S 2p 168.0 eV (18) peaks, indicating the presence of SO42−. The C 1s spectrum revealed a small peak of the C–C signal at 285.0 eV (1), which was speculated to be the carbon background in the air. When APTES was used to modify the silicon surface, the C 1s spectrum revealed peaks of the C–C signal (highest peak) at 285.0 eV (1), C–N signal at 286.6 eV (3), and COOR signal at 288.4 eV (7). The C–N bond is a part of the APTES structure, and COOR is a naturally occurring impurity in the APTES solution.21 In the N 1s spectrum, the APTES structure also produced an NH2 signal at 399.9 eV (14) and an oxide NH2/NH3+ signal at 401.9 eV (15). After modification with glutaraldehyde, significant peaks were observed at 287.7 eV (5) and 288.9 eV (6) in the C 1s spectrum, corresponding to C–N and HC=O, respectively. The signals were consistent with the composition of glutaraldehyde. The remaining spectra were similar to those obtained after APTES treatment. Next, we immobilized GST-PSGL-1 on the silicon surface and compared the two C 1s spectra obtained from the glutaraldehyde modification and GST-PSGL-1 immobilization steps. Because of the bond formation between the functional group H–C=O and GST-PSGL-1, the H–C=O peak at 288.9 eV (6) disappeared. In addition, the C 1s spectrum revealed peaks corresponding to H–C=O and C=O–N at 286.5 eV (3) and 288.0 eV (4), respectively. The O 1s spectrum exhibited peaks corresponding to N=O and C=O–OH at 530.3 eV (8) and 530.6 eV (9), respectively. The N 1s spectrum exhibited peaks corresponding to C–N and N=O at 397.6 eV (12) and 399.3 eV (13), 7



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respectively. Small peaks corresponding to C–S and unbonded disulfides at 160.6 eV (16) and 165.0 eV (17), respectively, appeared in the S 2p spectrum. These peaks were consistent with the amino acid composition of proteins. Finally, the surface was treated with PTS. The X-ray photoelectron spectroscopy (XPS) spectra were similar to those obtained for GST-PSGL-1 immobilization. PTS produced much fewer sulfur compounds than the sulfur that already exists in the GST-PSGL-1 structure; therefore, the S 2p spectra exhibited similar results.

Figure 2. Characterization of silicon surface by using XPS, SEM, and AFM. Confirmation of immobilized GST-PSGL-1 on the silicon surface. (a) XPS C 1s, O 1s, N 1s, and S 2p spectra verified the chemical components on the silicon surface. Chemical structures with binding energies are as follows: (1) C–C (285.0 eV), (2) H–C=O (286.5 eV), (3) C–N (286.6 eV), (4) 8


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C=O–N (288.0 eV), (5) C–N (287.7 eV), (6) H–C=O (288.9 eV), (7) COOR (288.4 eV), (8) N=O (530.3 eV), (9) C=O–OH (530.6 eV), (10) SO42− (532.5 eV), (11) SiOx (532.8 eV), (12) C– N (397.6 eV), (13) N=O (399.3 eV), (14) NH2 (399.9 eV), (15) NH2/NH3+ (401.9 eV), (16) C–S (160.6 eV), (17) unbonded disulfides (165.0 eV), and (18) SO42−, (168.0 eV). (b) Sulfated GSTPSGL-1 on the silicon surface obtained using SEM was examined after the addition of antisulfotyrosine and anti-mouse IgG conjugated with Au nanoparticles. SEM image of modified Au nanoparticles with a size of 10–12 nm (small white points) on the silicon surface is shown. (c) Image of AFM topography measurement. The silicon surface was washed with H2O2/H2SO4. Scan size: 1 µm × 1 µm; height: approximately 1.7 nm. GST-PSGL-1 protein was immobilized on the silicon surface. Scan size: 1 µm × 1 µm; average height: approximately 10.5 nm. The SAM procedure and the PTS reaction were followed by the sequential addition of antisulfotyrosine and anti-mouse IgG in preparation for the SEM examination. The SEM surface morphology of the SAM is presented in Figure 2(b). The anti-mouse IgG conjugated with Au nanoparticles (10–12 nm) recognized anti-sulfotyrosine. Therefore, sulfated GST-PSGL-1 was indirectly indicated by the Au nanoparticles (small white points) on the silicon surface. The results of negative control experiments with SEM are presented in Figure S3 in the supplementary data. Furthermore, AFM measurements before SAM formation revealed that the average background height of the silicon surface was 1.7 nm [Figure 2(c)]. When GST-PSGL-1 was immobilized onto the silicon surface, the average height increased to approximately 10.5 nm. The average height of the silicon surface is related to the dimensions of the protein. The PTS-induced PPI force results (see next section) provide indirect evidence of surface characterization.

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Antibody–antigen specific binding was used to establish a PTS-induced PPI measurement platform based on AFM. The spring constant of the cantilever was 0.16879 N/m. Details of the parameters are presented in Table S2 in the supplementary data. The SAM and AFM measurements revealed that the magnitude of a specific binding force was 2- to 16-fold higher than that of the nonspecific one.22-23 Therefore, we prepared a series of control experiments to confirm the SAM results. The substrate surfaces (groups), each treated with SAMs, were tested with an AFM probe immobilized with an antibody (anti-GST or anti-sulfotyrosine) (Figure 3). The anti-GST antibody confirmed the GST-PSGL-1 immobilization, and the anti-sulfotryosine confirmed PTS-induced PPIs. Groups 1–3 underwent a SAM process that lacked a certain reagent; the results revealed a low unbinding force of approximately 200–300 pN. Due to the absence of a reagent, GST-PSGL-1 could not be immobilized on the silicon surface. Therefore, the unbinding forces were for nonspecific binding. Group 4 underwent a blocking step with ethanolamine that prevented the nonspecific binding of antibodies with other proteins. GSTPSGL-1 can still be immobilized on the silicon surface without performing the blocking step; however, other unknown proteins may also adsorb onto the silicon surface, which may cause measurement inaccuracies. The results revealed that the unbinding force was the highest (approximately 1800–1900 pN) for group 4, which indicates that apart from the specific binding between GST-PSGL-1 and the antibody, the antibody also interacted with unknown proteins. Group 5 was a negative control for measuring PTS-induced PPIs. The PTS reaction of the silicon surface was untreated by the key enzyme (TPST); therefore, GST-PSGL-1 was still in its unsulfated form. The results revealed that the AFM probe for anti-GST and anti-sulfotyrosine exhibited unbinding forces of approximately 1346.3 and 240 pN, respectively. This indicates that a PPI force exists between anti-GST and GST-PSGL-1; however, no PPI force was observed 10


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between anti-sulfotyrosine and GST-PSGL-1. For groups 6 and 7, the GST-PSGL-1 protein and the PSGL-1 peptide were immobilized on the silicon surfaces, respectively. Then, the PTS reaction was performed on the silicon surfaces. Previous studies have demonstrated that the GST-PSGL-1 protein and the PSGL-1 peptide can be sulfated through the PTS reaction. Thus, sulfated GST-PSGL-1 was present on the silicon surface in group 6, allowing the anti-GST and anti-sulfotyrosine probes to recognize it. The unbinding forces were 1571.3 and 1601.8 pN, respectively. For group 7, only sulfated PSGL-1 peptide existed on the silicon surface; it was recognized by anti-sulfotyrosine. The unbinding forces of anti-GST and anti-sulfotyrosine were 245.8 and 1443.3 pN, respectively. The unbinding forces of PTS-induced PPIs between GSTPSGL-1 and anti-sulfotyrosine increased by 6.7-fold for specific binding. These results are consistent with those in the previous studies. Unbinding forces of up to 1500 pN were classified as specific binding forces. These results indirectly confirm the immobilization of the samples. The results of the GST-PSGL-1 protein and the PSGL-1 peptide confirmed that AFM measurement is not limited by protein size or source. Figure S4 presents the detailed AFM results for each group.

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Figure 3. AFM measurement of PTS-induced PPI forces between PSGL-1 and the antibody. Single molecular PPI force was confirmed by control experiments, in which the groups were distinguished by different modified steps of SAMs. Two types of antibody were immobilized onto the AFM probe for PPI force measurement. Groups 1–3 were used in background experiments and had no samples immobilized onto the silicon surface. Group 4 did not undergo the blocking step with ethanolamine that prevents the nonspecific binding of the antibody. Group 5 did not undergo the PTS reaction and was used as the negative control. Groups 6 and 7 were positive controls with immobilized GST-PSGL-1 protein and PSGL-1 peptide, respectively. The above table presents the specific forces between the groups and antibody. Detailed data are presented in Figure S4. 12


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In a biological system, the PPI force is usually weaker than the antibody–antigen interaction force. PPI force measurements require positive and negative controls to confirm their reliability. The antibody–antigen interaction force is the most effective positive control. Lack of TPST was used as the negative control. The actual PPI force value was calculated by subtracting the background force. Previous studies have demonstrated that for EV71 virus entry, sulfated PSGL1 and VP1 interaction is a critical step.22-23 PSGL-1 has three tyrosine residues at positions 46, 48, and 51; however, which of these residues is the major interaction position with VP1 is unknown. Therefore, we measured a series of PSGL-1 mutants with VP1 and an antibody to investigate the PTS-induced PPI forces. The statistical analysis of the average adhesion forces of anti-sulfotyrosine or VP1 probes and GST-PSGL-1 mutants are summarized in Figure 4. The detailed results are presented in Figures S5 and S6. The results revealed that anti-sulfotyrosine specifically binds with sulfated GST-PSGL-1. The unbinding forces of most mutants tend to decrease; however, for sulfated tyrosine-51 residues, the unbinding forces of (48F) and (46,48F) did not decrease. The results indicated that the mutant in the tyrosine-48 residue position did not affect the PPIs with anti-sulfotyrosine. In addition, the unbinding force of (46,48F) was 9.4-fold larger than that of (48,51F) and 6.8-fold larger than that of (46,51F). When the tyrosine-51 residue was replaced by phenylalanine, the unbinding force between GST-PSGL-1 and the antibody significantly decreased. The results indicated that sulfated tyrosine-51 residue is a major interaction position. The triple tyrosine mutation (46,48,51F) could not induce the PTS reaction, and the unbinding force indicated nonspecific binding. We also measured the PTSinduced PPIs on a sample using the VP1-modified AFM probe. The statistical analysis of the average adhesion forces of the VP1 probe and GST-PSGL-1 mutants is summarized in Figure 4 (open column). The results revealed that the unbinding forces of proteins were relatively lower. 13



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The unbinding force of (46,48F) was 4.5-fold larger than that of (48,51F) and 5.4-fold larger than that of (46,51F). When the tyrosine-51 residue was replaced, the unbinding force between sulfated GST-PSGL-1 and VP1 significantly decreased. These results are similar to those for anti-sulfotyrosine; however, the unbinding forces of VP1 were only approximately one-third of those of anti-sulfotyrosine. The three tyrosines are generally reported as potential sulfation sites and may contribute to the specific binding of its ligand,18 such as P-selectin, anti-sulfotyrosine antibody, and VP1 protein. However, whether these ligands recognized all or part of the sulfated tyrosines was unclear. Our study highlighted that only the sulfation at tyrosine-51 significantly induced PPI. However, notably, mutation at tyrosine-46 also plays a role in PPI induction. Mutation of this tyrosine to phenylalanine significantly reduced PPI with both anti-sulfotyrosine antibody and VP1 protein. Our recent study indicated that this tyrosine could not be sulfated through TPST-catalyzed PTS (unpublished data). We propose that structural factor instead of PST of tyrosine-46 affects the PPIs observed in this study.

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Figure 4. PPI force between GST-PSGL-1 mutants and antibody/VP1. Single molecular PPI force between protein partners is shown. Bars indicate the unbinding force between GST-PSGL1 mutants immobilized on the silicon surface and AFM probe. The antibody and VP1 were immobilized onto the AFM probe for PPI force measurement. Unbinding force values were obtained by averaging the measurement data of the given points. Unbinding force was measured at least at three points to confirm PTS-induced PPI forces. Each bar had the background force removed, indicating real interaction force. Detailed data are presented in Figures S5 and S6. In conclusion, we determined that sulfation at tyrosine-51 of PSGL-1 was mainly responsible for TPST-induced PPI through the preparation of a series of purified recombinant GST fusion proteins for AFM measurement. The results indicate that AFM can be used to measure the PTSinduced PPI force of high-purity homogenous samples of recombinant proteins. Moreover, AFM was applied to measure the GST-PSGL-1 mutant PPI force with an antibody and VP1. The results indicate that irrespective of the antibody or VP1 AFM probe used, the sulfated tyrosine51 residue of GST-PSGL-1 was the major PPI position. Therefore, the tyrosine-51 residue of GST-PSGL-1 plays a critical function in PTS-induced PPIs. According to the PPI force results, this AFM platform can be applied to protein and peptide measurements to specifically determine the interaction site and force. Supporting Information. Experimental methods details, additional Tables and Figures. Acknowledgement This work was supported by the Ministry of Science and Technology, Taiwan (106-3114-E-009001; 105-2311-B-009-001). We acknowledge the facilities and the scientific and technical assistance of the industrial technology research institute (ITRI).

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(22) Lu, L.Y.; Chen, B.H.; Wu, J.Y.; Wang, C.C.; Chen, D.H.; Yang, Y.S. Implantation of posttranslational tyrosylprotein sulfation into a prokaryotic expression system. Chembiochem. 2011, 12, 377379. (23) Chen, B.H.; Wang, C.C.; Lu, L.Y.; Hung, K.S.; Yang, Y.S. Fluorescence assay for protein posttranslational tyrosine sulfation. Anal Bioanal Chem. 2013, 405, 1425-1429. (24) Min, H.; Girard-Lauriault, P.-L.; Gross, T.; Lippitz, A.; Dietrich, P.; Unger, W. E. S. Ambientageing processes in amine self-assembled monolayers on microarray slides as studied by ToF-SIMS with principal component analysis, XPS, and NEXAFS spectroscopy. Anal Bioanal Chem. 2012, 403, 613623. (25) Lv, Z.; Wang, J.; Chen, G.; Deng, L. Probing Specific Interaction Forces Between Human IgG and Rat Anti-Human IgG by Self-Assembled Monolayer and Atomic Force Microscopy. Nanoscale Res Lett. 2010, 5, 1032-1038. (26) Kaur, J.; Singh, K. V.; Schmid, A. H.; Varshney, G. C.; Suri, C. R.; Raje, M. Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface. Biosens Bioelectron. 2004, 20, 284-293.

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TOC 127x89mm (300 x 300 DPI)


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Figure 1 127x106mm (300 x 300 DPI)



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Figure 2 177x126mm (300 x 300 DPI)


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Figure 3 135x129mm (300 x 300 DPI)



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Figure 4 135x106mm (300 x 300 DPI)


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Specific Unbinding Forces Between Mutated Human P-Selectin

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